Cell adhesion to extracellular matrix (ECM) initiates multiple signaling cascades including activation of focal adhesion kinase (FAK) family kinases and Rho family GTPases that regulate the organization of actin cytoskeleton and changes in gene expression. Rho family GTPases, particularly Rho, Rac, and CDC42, have emerged as central coordinators of a variety of cellular processes including actin cytoskeletal organization. FAK family kinases including FAK and proline-rich tyrosine kinase 2 (PYK2) play important roles in regulating cell migration. The mechanisms by which PYK2 and FAK participate in and regulate actin cytoskeletal organization remain largely unknown. How FAK family kinases cross talk with Rho family GlPases to coordinately organize the complex signaling required for actin cytoskeletal organization and cell migration is a central question of this proposal. In preliminary studies, we found that PYK2 and FAK are upstream regulators of CDC42 and RhoA. Using yeast two-hybrid system, we identified a novel PYK2 and FAK interacting protein, PSGAP (for PH domain and SH3 domain containing RhoGAP protein), which may be a crucial signaling protein for PYK2 and FAK to cross talk with Rho family proteins. Based on these preliminary results, we hypothesize that PYK2 and FAK regulate Rho family GTPases via PSGAP, to organize actin cytoskeleton and to modulate cell migration. To test this hypothesis, we will: 1) determine the role of PSGAP in integrin- or FAK-mediated transient inactivation of Rho, cell spreading, and cell migration; 2) determine the role of PSGAP in integrin- or PYK2-mediated activation of CDC42 and actin cytoskeletal reorganization; 3) investigate the mechanisms by which PSGAP is regulated. The proposed research will start to piece together a signaling pathway by which FAK and PYK2 coordinately regulate actin cytoskeletal organization and cell migration. The resulting data will provide insight into mechanisms by which integrin regulates cell adhesion and migration. Such knowledge is essential for understanding the mechanisms involved in abnormal adhesion that leads to pathological states, such as tumor metastasis.